Flaviviruses are small, enveloped, positive-strand RNA viruses that are of concern in many medical and veterinary settings throughout the world. West Nile virus (WN, or WNV), for example, which is a member of the flavivirus family, is the causative agent of WN encephalitis, an infectious, non-contagious, arthropod-borne viral disease (In Virology, Fields (ed.), Raven-Lippincott, New York, 1996, pp. 961-1034). The virus has been found in Africa, western Asia, the Middle East, the Mediterranean region of Europe, and, recently, in the United States. Mosquitoes become infected with the virus after feeding on infected wild birds, and then transmit the virus through bites to humans, birds, and animals, such as horses, sheep, cattle, and pigs.
West Nile virus is an emerging infectious disease. West Nile virus was first isolated in Uganda in 1937. Today it is most commonly found in Africa, West Asia, Europe, and the Middle East. However, it made its first recognized appearance in the United States in 1999. By 2004, the virus had been found in birds and mosquitoes in every state except Alaska and Hawaii.
Other well-known diseases caused by flaviviruses include yellow fever, Japanese encephalitis, Dengue, and Saint Louis encephalitis. Flavivirus infections are commonly transmitted by ticks and/or mosquitoes.
The primary hosts for West Nile are only mosquitoes and birds. Other animal species, such as humans, and animals, such as horses, sheep, cattle, and pigs, and the like are thought only to be incidental hosts that become infected when an infected female mosquito bites the incidental host.
People who contract West Nile virus usually experience only mild symptoms including fever, headache, body aches, skin rash, and swollen lymph glands. If West Nile virus enters the brain, however, it can cause life-threatening encephalitis or meningitis. Life-threatening cases primarily occur in the elderly. Recent studies have shown that West Nile virus can be transmitted through blood transfusions and organ transplants. Some health experts also believe it is possible for West Nile virus to be transmitted from a mother to her unborn child, and through breast milk.
There are many development projects for West Nile virus vaccine approaches, including live chimeric vaccines (which combine genes from more than one virus into a single vaccine), naked DNA vaccines, and vaccines containing cocktails of individual West Nile proteins, and the like. However, there is no approach making use of an inactivated chimeric vaccine.
Flavivirus proteins are produced by translation of a single, long open reading frame to generate a polyprotein, which undergoes a complex series of post-translational proteolytic cleavages by a combination of host and viral proteases to generate mature viral proteins. The virus structural proteins are arranged in the polyprotein in the order C-prM-E, where “C” is capsid, “prM” is a precursor of the viral envelope-bound M (membrane) protein, and “E” is the envelope protein. These proteins are present in the N-terminal region of the polyprotein, while the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are located in the C-terminal region of the polypeptide.
In 2003, human clinical trials of a West Nile live, attenuated virus vaccine were begun by Acambis (Cambridge, Mass.). The Acambis live, attenuated vaccine is based on a vaccine already used for preventing yellow fever, a disease caused by a different flavivirus.
One Acambis live, attenuated vaccine contains genes from two different viruses, yellow fever and West Nile, and is an example of a chimeric virus. This Acambis live, attenuated vaccine comprises a Yellow Fever virus with a few genes replaced with genes for surface proteins of West Nile virus.
Details of making this live, attenuated chimeric Acambis vaccine are provided, for example, in U.S. Pat. Nos. 6,962,708 and 6,696,281 and Chambers et al., J. Virol. 73:3095-3101, 1999, which are each incorporated by reference herein in their entirety. Further methods of use and diagnostics for the Acambis live, attenuated chimeric vaccine are provided in U.S. Pat. Nos. 6,682,883 and 6,878,372, which are each incorporated by reference herein in their entirety.
The results of such live, attenuated vaccines have proven successful and trials continue. However, certain risks may accompany the use of a live, attenuated virus vaccine. These risks are even more pronounced for immuno-compromised subjects, the elderly subjects, pregnant subjects, and other subjects with a weakened or stressed immune system. Quite often, live, attenuated virus vaccines have been demonstrated to be either under-attenuated (cause disease) or over-attenuated (fail to immunize). It is also possible for an optimally-attenuated live virus vaccine to revert to a virulent (disease-causing) form through mutation. However, it should be noted that the YF-WN from Acambis has shown no indication of reversion to virulence. There are additional concerns with live attenuated vaccines. For example, live Dengue viruses are also sensitive to heat, making it difficult and costly to maintain the vaccine in some tropical and subtropical countries where the vaccine may be needed most. Accordingly, a vaccine is needed in the art for safely treating and/or preventing flavivirus infections, such as West Nile, in subjects with these or other similar risks. Particularly for those who are immune compromised or other subjects most at risk.
However, the state of the art is that an inactivated chimeric virus vaccine is undesirable and would not be effective. U.S. Pat. No. 6,432,411 reported that efforts to make killed flavivirus vaccines have met with limited success. Primarily the studies were limited by the inability to obtain adequate virus yields from cell culture systems. Virus yields from insect cells are generally in the range of 104 to 105 pfu/ml, well below the levels necessary to generate a cost-effective killed vaccine. Yields from mammalian cells including LLC-MK2 and Vero cells were higher, but the peak yields, approximately 108 pfu/ml from a unique Vero cell line, are still lower than necessary to achieve a truly cost-effective vaccine product.
Accordingly, the art teaches away from the use of inactivated flaviviruses as viable vaccine candidates. Moreover, there is no teaching of an inactivated chimeric vaccine for treating or preventing any flavivirus infection.